Development of many vertebrate tissues involves long-range cell migrations. In most cases, these migrations have been inferred from analysis of single time points and the migration process has not been directly observed and quantitated in real time. In the mammalian adult thymus, immature CD4+ CD8+ double-positive (DP) thymocytes are found in the outer cortex, whereas after T cell antigen receptor (TCR) repertoire selection, CD4+ CD8- and CD4- CD8+ single-positive (SP) thymocytes are found in the central medulla. Here we have used two-photon laser-scanning microscopy and quantitative analysis of four-dimensional cell migration data to investigate the movement of thymocytes through the cortex in real time within intact thymic lobes. We show that prior to positive selection, cortical thymocytes exhibit random walk migration. In contrast, positive selection is correlated with the appearance of a thymocyte population displaying rapid, directed migration toward the medulla. These studies provide our first glimpse into the dynamics of developmentally programmed, long-range cell migration in the mammalian thymus.